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Faculty Publications - , Merchandising Textiles, Merchandising and Fashion Design, and Fashion Design Department of

2013

Structure and Properties of Cocoons and Fibers Produced by atlas

Narendra Reddy University of Nebraska - Lincoln, [email protected]

Yi Zhao University of Nebraska–Lincoln, [email protected]

Yiqi Yang University of Nebraska-Lincoln, [email protected]

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Reddy, Narendra; Zhao, Yi; and Yang, Yiqi, "Structure and Properties of Cocoons and Silk Fibers Produced by " (2013). Faculty Publications - Textiles, Merchandising and Fashion Design. 32. https://digitalcommons.unl.edu/textiles_facpub/32

This Article is brought to you for free and open access by the Textiles, Merchandising and Fashion Design, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications - Textiles, Merchandising and Fashion Design by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Published in Journal of Polymers and the Environment 21 (2013) 21:16–23; doi: 10.1007/s10924-012-0549-8 Copyright © Springer Science+Business Media New York. Used by permission. Published online November 8, 2012

Structure and Properties of Cocoons and Silk Fibers Produced by Attacus atlas

Narendra Reddy,1 Yi Zhao,1 and Yiqi Yang1, 2, 3

1. Department of Textiles, Merchandising & Fashion Design, University of Nebraska–Lincoln, 234 HECO Building, East Campus, Lincoln, NE 68583-0802, USA 2. Department of Biological Systems Engineering, University of Nebraska–Lincoln, 234 HECO Building, East Campus, Lincoln, NE 68583-0802, USA 3. Nebraska Center for Materials and Nanoscience, University of Nebraska–Lincoln, 234 HECO Building, East Campus, Lincoln, NE 68583-0802, USA Corresponding author — Yiqi Yang, email [email protected]

Abstract Introduction

Silk fibers in the three layers of Attacus atlas (A. at- Silk fibers and protein based materials, in general, are las) cocoons have morphological structure and ten- preferred for biomedical, and biotechnology indus- sile properties similar to that of silk. tries [1–5]. In addition to the common Bombyx mori , Attempts are being made to produce silk for com- silks from several other sources such as spider silks and mercial applications from cocoons of relatively un- non-domesticated silk worms such as Antheraea pernyi are known wild due to the unique properties of being studied for potential use in various applications [6, the fibers and as a source of income and employ- 7]. For instance, the exceptional tensile strength of spider ment. In this research, A. atlas cocoons were used to silk has been well documented and several attempts have study the chemical composition, morphology, phys- to made to produce regenerated spider silk [8]. Similarly, ical structure and tensile properties of the silk fibers it has been found that fibroin from wild silks has better in the cocoons and ability of the fibers to support potential for medical applications compared to fibroin the attachment and proliferation of mouse fibro- from B. mori silk [9]. Recent studies also suggest that, con- blast cells. It was found that A. atlas cocoons consists trary to popular belief, silk sericin also has the potential of outer, intermediate and inner layer with average for medical applications [9, 10]. Although a wide variety breaking tenacity of 4.1, 4.3, and 3.6 g/den, respec- of insects have been known to produce silk containing co- tively similar to that of B. mori silk (4.3–5.2 g/den). coons, only a few non-mulberry silks have been studied The heavier cocoons, less restrictive rearing condi- for their structure, properties and potential applications. tions and good properties of the fibers compared to Insects belonging to the family are one of the B. mori silk makes A. atlas a potential alternative to largest group of insects known to produce cocoons con- common silks for commercial scale silk production. taining silk [11]. Among the numerous saturniidae in- A. atlas fibers had about 80 % higher optical densi- sects, Antheraea mylitta (Indian tasar), Antheraea pernyi ties of cells and extensive growth of F-actin com- (Chinese tasar), and Philosamia ricini (Eri) have been ex- pared to B. mori silk fibers. tensively studied for their structure, properties and ap- plications [12, 13]. Silk from these wild insects is being Keywords: Wild silks, Attacus atlas, Biocompatibil- processed for commercial applications. However, the ity, Tensile properties, Bombyx mori common non-mulberry fibers are much coarser

16 P r o p e r t i e s o f C o c o o n s a n d S i l k F i b e r s P r o d u c e d b y A t t a c u s a t l a s 17 than the B. mori silk [14]. Therefore, the wild silk fibers Degumming have limited applications compared to B. mori silk fibers. In addition to the three saturniidae insects A. mylitta, A. The A. atlas cocoons were separated into three layers by pernyi, and P. ricini, other insects also belonging to the sa- hand. The three layers were separately degummed us- turniidae family such as Eupackardia callata, ing ethylenediamene and sodium carbonate. Cocoon lebeau, Hyalophora gloveri, Actias luna, Coscinocera hercu- layers were treated in 10 % ethylenediamine and 0.5 % les, Antheraea oculea, and Copaxa multifenestrata also pro- sodium carbonate for 1 h at 80 °C with a solution to co- duce cocoons containing silk. Fibers from C. hercules were coon ratio of 20:1. Later, cocoons were washed in water found to have properties similar to that of B. mori silk and retreated in the ethylenediamine and sodium car- whereas fibers from C. multifenestrata were much coarser bonate solution at 80 °C for 15 min to complete the de- and had low breaking tenacity and breaking elongation gumming and obtain loose fibers. Degummed silk was compared to B. mori silk [15]. Likewise, Hylaphora cecro- thoroughly washed in water and dried under ambient pia cocoons were reported to consist of three layers with conditions. properties similar to that of B. mori silk [16]. However, silk produced by bag worms was found to be weak and Morphology with considerably different amino acids than those found in common silks [17]. Images of the three different layers of A. atlas cocoons In addition to obtaining fibers with unique proper- were collected using a digital camera. Surface features ties, utilizing cocoons from wild insects for commercial of the different layers in the cocoons and the longitu- silk production will provide economic benefits. Since the dinal and cross-sectional features of the degummed fi- wild insects are located in forest and tribal areas, devel- bers were observed using a variable pressure scanning oping silks from the wild insects will provide income to electron microscope (VP-SEM). Samples were sputter the indigenous people. Therefore, efforts are being made coated with gold palladium and observed in the SEM at to rear wild silks for commercial silk production [18, 19]. a voltage of 20 kV. Attacus atlas belongs to the saturniidae family which includes the common wild silks currently used for com- Composition mercial silk production. Attacus atlas insects are primar- ily found in the tropical forests and are considered to The type and percentage of amino acids in the A. at- the largest insects based on their wing span las fibers was determined using a Hitachi L-8800A which exceeds 65 square inches [20]. These insects pro- amino acid analyzer. Norleucine was used as the inter- duce silk containing cocoons and the color of the co- nal standard for both the samples and the control for coons varies from silver to black depending on the host amino acid analysis. Samples were first evaporated to plant. Attacus atlas cocoons have been processed to de- dryness in a speedvac and later hydrolyzed in liquid velop yarns and fabrics. In another report, tensile prop- 6 N HCl under argon atmosphere. After 20 h of hydro- erties of the A. atlas silk fibers submerged in liquid me- lysis at 110° C, the samples were evaporated to dry- dia have also been studied [21]. It was reported that ness, then redissolved in 200 μL of 0.02 N HCl. Fifty water disrupted the protein–protein hydrogen bonds microliters was injected automatically onto the Hit- whereas ethanol enhanced the protein–protein hydro- achi Amino Acid Analyzer to determine the amino gen bonds. Significant differences in tensile properties acid type and content. In data analysis, correction was of A. atlas and spider silk were observed even though made to the amount of the internal standard, to mini- they had similar amino acid composition. The previous mize dilutional errors. research by Perez-Rigueiro was focused on understand- ing the tensile properties of the A. atlas silk fibers in liq- Physical Structure uid media [21]. In this research, we have studied the properties of the A. atlas cocoons and the silk fibers from Physical structure of the A. atlas fibers was determined the three layers in A. atlas cocoons have been character- in terms of % crystallinity and peak positions using a ized for their structure and properties in comparison to Rigaku D-max/BΘ/2Θ X-Ray diffractometer (Rigaku B. mori and common wild silks. Americas, Woodlands, TX) with Bragg–Brentano para- focusing geometry, a diffracted beam monochromator, Experimental and a copper target X-ray tube set to 40 kV and 30 mA. Fibers were powdered and compressed to form pellets Materials for the X-ray measurements. Powdered samples were used for the X-ray analysis to eliminate any preferred Attacus atlas cocoons were supplied by Reiman Gardens orientation of the crystals to the fiber axes. This is a com- in Ames, Iowa. Chemicals used for the study were pur- mon practice in obtaining X-ray diffraction data for fi- chased from VWR international (Bristol, CT). bers. Diffraction intensities were collected for 2θ val- 18 R e d d y , Z h a o , & Y a n g i n J o u r n a l o f P o l y m e r s a n d t h e E n v i r o n m e n t 21 (2013) ues ranging from 5 to 40°. The % crystallinity of the fiber Fibers after cell culture were also observed in a Con- was obtained by integrating the area under the crystal- focal laser scanning microscope (Olympus, Model: line peaks after subtracting the background and air scat- FV500) to detect the extent of cell growth and spread- ter using the program MICROCAL ORIGIN. ing of the cytoskeleton. For the confocal study, the cells were fixed using 4 % paraformaldehyde and then stained with the fluorescent dyes (Phallodin 633 and Tensile Properties and Moisture Regain Hoechst 33342) to stain the cell and nuclei in red and blue, respectively. Samples were conditioned for at least 24 h under the standard testing conditions of 21 °C and 65 % relative humidity before testing. Fineness of the fibers was Results and Discussion calculated in terms of denier (weight in grams per 9,000 m of the fibers) by weighing a known length of Construction of the Cocoon the fibers. The tensile properties of the fibers from the outer, intermediate and inner layers were determined Attacus atlas cocoons used in this research had an aver- separately on an Instron tensile tester using a gauge age weight of 1.21 g, much higher than that of B. mori length of 1 inch and cross head speed of 18 mm/ (640 milligrams) and P. ricin (840 mg) cocoons but lower minute. At least 50 fibers from ten different cocoons than that of the A. mylitta (3.4 g) cocoons [12, 13]. The A. were tested and the average and standard deviations atlas cocoons are constructed in three distinct layers. The were obtained. Moisture regain of the fibers was de- outer layer was loosely attached and was paper-like as termined according to ASTM standard method 2654 seen from Figure 1a and had a thickness of 0.5 ± 0.2 mm using standard conditions of 21 °C and 65 % relative and accounted for approximately 23 % of the total humidity. weight of the cocoon. In comparison, middle or interme- diate layer (Figure 1b) with a thickness of 0.6 ± 0.2 mm formed the bulk of the cocoons and accounted for 43 % Cell Culture and Biocompatibility of the weight of the cocoon. The intermediate layer con- tains most of the fibers and was loosely connected to Ability of A. atlas fibers to support the attachment and the outer layer but very tightly connected to the inner growth of mouse fibroblast cells was studied in compar- layer. Innermost layer seen in Figure 1c accounted for ison to the common B. mori silk. Silk fibers were com- approximately 34 % of the weight of the cocoon and had pressed into a sheet and sterilized in an autoclave at an average thickness of 0.20 ± 0.09 mm. As seen from 120 °C. Samples were accurately weighted and placed Figure 1c, the size of the layers decreased progressively in a 24-well culture plate and incubated with NIH3T3 from the outer layer to the inner layer. It is reported that mouse fibroblast cells at a concentration of 1.5 × 105 the decreases its size as it builds the cocoons cells mL−1 DMEM media containing 4 mM l-glutamine, from outside to inside making the cocoons compact 10 % calf serum, and 1.0 % of 104 μg/mL penicillin/ with the inner most layer being the smallest. streptomycin were added into each well and the plates were incubated at 37 °C and 5 % CO2 up to 11 days. Me- Effect of Degumming dia was refreshed every 3 days. After the desired length of incubation, the samples were transferred to a new Degumming resulted in removal of the gums (sericin) culture plate and the unattached cells were removed by and disintegration of the cocoons into lustrous fibers. rinsing with phosphate buffered saline. Metabolic ac- In addition to sericin, wild silks cocoons are mineral- tivities of the cells on the silk fibers were measured us- ized and contain considerable amounts of calcium oxa- ing a MTS assay. To perform the assay, about 0.5 mL per late crystals that affect degumming and the properties well of 20 % MTS solution in DMEM media was added of the cocoons. It has been reported that treating with into each well and incubated for 3 h. After 3 h, 150 μL ethylenediamine is effective in removing calcium oxa- of DMEM was collected into a 96-well plate and the ab- late from the cocoon. Among the three layers of the co- sorbance of the solution was read on a Mutiwell plate coons, the outer layer of A. atlas cocoons had the highest reader (Thermoscientific Model: Multiskan) at a - wave degumming loss of 22 % followed by the inner and in- length of 490 nm to obtain the optical densities. Blank termediate layers with degumming loss of 10 and 7.3 %, samples without any cells were also tested and used as respectively. Relatively stronger conditions are required control. At least four samples were tested for each time to degum wild silk fibers compared to B. mori silk fibers point and the experiments were repeated twice. The av- due to the chemical interactions between wild silk ser- erage optical density of the cells with ± one standard de- icins and inorganic components and the minerals pres- viation are reported. ent in the cocoons [22]. B. mori silk fibers are reported to P r o p e r t i e s o f C o c o o n s a n d S i l k F i b e r s P r o d u c e d b y A t t a c u s a t l a s 19

sericin content in the B. mori cocoons varied from about 30 % in the outer layer to about 20 % in the inner most layer [23]. Although sericin needs to be removed from silk fibers to make the fibers lustrous, sericin proteins are found to have unique properties and used for ap- plications in various areas. Sericin resists oxidation, has antibacterial and anti-UV resistant properties [22, 24]. Silk sericin has been used for medical, pharmaceutical and cosmetic industries [24, 25].

Morphology of the Layers in the A. atlas Cocoons

SEM images of the three layers of A. atlas cocoons are shown in Figure 2. As seen from the figure, the outer layer (Figure 2a) has a relatively open construction com- pared to the intermediate (Figure 2b) and inner layers (Figure 2c). The inner layer is very compact and has fi- bers much closer to each other than the fibers in the outer and intermediate layers as seen from Figure 2c. As seen from Figs. 2a to c, the A. atlas caterpillar extrudes two adjacent fibers simultaneously. The two adjacent -fi bers are attached to each other by glues in the interme- diate and inner layers but the outer layer has fibers that are not as extensively attached to each other as fibers in the other two layers. Inner layer also had fibers with slightly lower diameters than the fibers in the outer and intermediate layers. Average diameter of a single fiber in the inner layer was 17 ± 5 μm and those in the outer and intermediate layer was 19 ± 4 and 20 ± 4 μm, respec- tively. It has been reported that the filaments in A. at- las consist of microfibrils of about 1 μm in diameter and embedded in a soft matrix [26].

Composition

Silk produced by A. atlas has considerably different composition of amino acids than the common silks as seen from Table 1. Among the major amino acids, A. at- las has more than twice the tyrosine, about 53 % higher alanine but about 50 % lower glycine and 56 % lower serine content than B. mori silk. However, the alanine and glycine content in A. atlas silk is similar to that of the wild silks. The glycine/alanine ratio for A. atlas silk Figure 1. Structure of the A. atlas cocoons. a shows an intact cocoon, b reveals the fiber (intermediate) layer after the outer is 0.5, much lower than that of mulberry (1.5) and the most layer has been cut open and c shows the three layers common wild silks (0.8). Amount of glycine and alanine (outer, intermediate and inner layer) in the cocoons. determines the crystallographic form of the proteins [12, 13]. The lower ratio glycine/alanine in A. atlas silk sug- gests that A. atlas silk could have a considerably differ- have sericin content ranging from 20 to 30 % and mild ent crystallographic structure compared to mulberry alkali at 90 °C and degumming without ethylenedi- and wild silks. A. atlas silk has much higher amounts amine or other chelating agents are normally adequate of hydrophobic amino acids (67 %) compared to the hy- for B. mori silk [22]. It has also been reported that the drophilic amino acids (23 %) indicating that the A. at- sericin content in cocoons progressively decreases from las silk may have lower moisture absorption and lower the outside layers to the inside layers similar to that ob- sorption of dyes and chemicals if the crystal structures served for the A. atlas cocoons in this research [23]. The and % crystallinity are similar. 20 R e d d y , Z h a o , & Y a n g i n J o u r n a l o f P o l y m e r s a n d t h e E n v i r o n m e n t 21 (2013)

Table 1. Comparison of the amino acid composition of the silk from the fiber layer in A. atlas cocoons with B. mori and three varieties of common wild silks Amino acids % Amino acids A. atlas B. mori A. mylitta A. pernyi P. ricini Alanine 45.0 29.4 34.1 34.7 36.3 Tyrosine 11.4 5.2 6.8 5.1 5.8 Glycine 22.2 44.6 27.7 28.4 29.4 Serine 6.8 12.1 9.9 9.1 8.9 Aspartic acid 3.2 1.3 6.1 5.0 3.9 Arginine 2.2 0.5 5.0 4.7 4.1 Glutamic acid 3.6 1.8 1.3 1.4 1.3 Histidine 2.2 0.1 0.8 0.7 0.8 Data for B. mori and the wild silks are from [11–13]

acids compared to 22.2, 45 and 6.8 %, respectively in this research. However, the total glycine, alanine and ser- ine in A. atlas previously reported and found in this re- search are similar at 74 and 67.5 %, respectively. In the previous study by Perez-Rigueiro, it was suggested that glycine, alanine, serine and threonine exist in the crystalline region and all other amino acids exist in the amorphous region. The ratio of amino acids in the crys- talline and non-crystalline regions was called the disor- der ratio and related to the properties of the fibers [21]. The variations in the amino acid contents of A. atlas silk in the previous report and current research should be due to habitat of the insects, rearing conditions and dif- ferences in the methods used to determine the amino acids.

Physical Structure

X-ray diffractogram of A. atlas fibers has been com- pared to B. mori silk fibers in Figure 3. A. atlas fibers had two strong distinct diffracting peaks at 17 and 20° corresponding to the reflections from the 002 and 201 planes whereas B. mori silk had a single diffracting peak at about 20.6° corresponding to the 201 plane reflection [12, 13]. However, other non-mulberry silks also showed the presence of two diffracting peaks at 17 and 20°. It has been suggested that the nature of crystals in silk is dependent on the alanine/glycine ratio. The higher ala- nine content provides a better crystallographic form for the A. atlas silk compared to B. mori silk. The % crystal- Figure 2. SEM images of the outer (a), intermediate (b) and in- ner (c) layers in the A. atlas cocoons before degumming. The linity of A. atlas fibers was 32.8 % close to the high end white particles in the cocoons are the calcium oxalate crystals. reported for B. mori (20–41 %) silk fibers [12, 13].

Morphology of the Fibers A previous report studying the properties of A. atlas silk fibers immersed in liquid media has reported con- Morphology of the A. atlas fibers after degumming are siderably different amino acid composition [21]. In that shown in Figs. 4 and 5. Longitudinally, the fibers have report, A. atlas silk was reported to contain 40.7 % gly- a clean and smooth surface as seen from Figure 4. Fig- cine, 18 % alanine and 8.8 % serine as the major amino ure 5a and b reveals that the fibers are flat and rib- P r o p e r t i e s o f C o c o o n s a n d S i l k F i b e r s P r o d u c e d b y A t t a c u s a t l a s 21

Figure 3. X-ray diffractogram of A. atlas fibers compared to B. mori silk fibers. bon-like and have a solid cross-section. The morphol- ogy of the A. atlas fibers is similar to that of the wild silk but different than B. mori silk that have a triangular cross-section.

Tensile Properties

The fineness, tensile properties and moisture regain of the silk fibers from the three layers of A. atlas cocoons are compared with that of B. mori and two common wild silks in Table 2. As seen from the table, fibers from the outer layer of A. atlas cocoons are coarser than the fibers from the intermediate and inner layer. However, the A. Figure 5. SEM images a, b of A. atlas fibers showing the rectan- atlas silk fibers from all the three layers are coarser than gular to circular cross-section and smooth surface. B. mori silk fibers but much finer than A. mylitta and P. ricini silk fibers. Although the inner layer of A. atlas co- coons contains the finest fibers, the breaking tenacity of the fibers in the inner layers (3.6 g/den) was lower than layer were similar to that of B. mori silk and better than the fibers in the outer and fiber layer. The breaking te- that of P. ricini silk. The average breaking elongation nacity of the fibers from the outer and intermediate (15–17.3 %) of the A. atlas fibers was similar to that of B. mori silk fibers but lower than that of the wild silks whereas the Young’s modulus of the A. atlas silk fibers was lower than that of B. mori but higher than that of P. ricni silk fibers as seen from the stress–strain curve in Figure 6. The variations in the tensile properties of the silk fibers from different insects should be due tothe differences in the composition, % crystallinity and ar- rangement of the crystals in the fibers. Tensile properties of A. atlas silk submerged in liq- uid media was previously reported [20]. It was reported that immersion in water greatly reduced the elastic modulus but immersion in ethanol increased the mod- ulus. Although A. atlas fibers have different amino acid composition compared to that of B. mori silk fibers, they have similar mechanical properties. However, the A. at- las fibers have considerably different mechanical prop- erties than spider silk although spider silk and A. atlas Figure 4. SEM image of A. atlas fibers showing the smooth sur- have similar amino acid composition [21]. It has also face of the fibers after degumming. been suggested that the tensile failure mechanism of A. 22 R e d d y , Z h a o , & Y a n g i n J o u r n a l o f P o l y m e r s a n d t h e E n v i r o n m e n t 21 (2013)

Table 2. Tensile properties of the silk fibers from the three layers of the A. atlas cocoons compared to a range of tensile properties for B. mori, A. mylitta and P. ricini silks Fiber A. atlas Outer Intermediate Inner B. mori A. mylitta P. ricini

Fineness, denier 2.7 2.0 1.7 0.4–1.1 4.7–10.7 2.3–3.6 Strength, g/den 4.1 ± 1.5 4.3 ± 0.8 3.6 ± 0.6 4.3–5.2 3.9–4.5 1.9–3.5 Elongation, % 17.3 ± 8.6 18.7 ± 9.3 15.0 ± 6.4 10.0–23.4 26–39 24–27 Modulus, g/den 53 ± 18 48 ± 18 60 ± 12 84–121 66–70 29–31 Moisture regain, % 11.6 12.0 12.4 8.5–9.9 10.5 10.0

Data for B. mori and the wild silks are from [11–13]

Figure 6. Stress-strain curves of fibers from the three layers Figure 7. Optical densities per unit weight of the A. atlas and in A. atlas cocoons compared to B. mori and the common wild B. mori fibers based on the MTS assay after culture at 37 °C and silks A. mylitta and P. ricini. 5 % CO2 . atlas fibers involves the pulling of micofibrils from the along the length of the fibers. Overall, the cell culture matrix, similar to that observed in composites [26]. The studies demonstrate that A. atlas fibers are biocompat- moisture regain of the A. atlas silk fibers was higher than ible and would be suitable for tissue culture and other that of the other silk fibers in Table 2. Overall, the tensile medical applications. properties of the A. atlas fibers are similar to that of B. mori silk than the common wild silks. Conclusions Cell Culture Attacus atlas produces considerably heavier cocoons Attacus atlas fibers showed considerably higher cell den- with three distinct layers. The intermediate layer formed sities from the MTS assay compared to the B. mori silk fi- the bulk (43 %) of the cocoons whereas the outer and in- bers. Cells on the A. atlas fibers reached confluence af- ner layers accounted for 23 and 34 %, of the weight of ter 5 days of culture compared to 7 days for the B. mori the cocoon, respectively. Degumming was necessary to silks. As seen from Figure 7, the cell density on the A. obtain fibers from the cocoons and the extent of degum- atlas fibers was higher on all days of culture compared ming loss decreased from the outer to the inner layers. to the B. mori fibers. At their respective days of conflu- The amino acid composition in the A. atlas fibers was ence, the A. atlas fibers had nearly 80 % higher cell den- found to be considerably different than the amino acid sities per unit area of the fibers suggesting that the A. composition in B. mori silk fibers. Fibers in the outer atlas fibers were more conducive to cell attachment and layer of the cocoons were relatively coarser than the fi- growth. Confocal image in Figure 8 showed that the bers in the intermediate and inner layer. Breaking tenac- cells had extensive spreading of the cytoskeleton (red) ity (3.6–4.3 g/den) of the fibers from the three layers of and the presence of numbers of cells (cell nuclei in blue). A. atlas cocoons was similar to that of B. mori silk but The left panel in Figure 8 shows that the cytoskele- much higher than that of the common wild silks. Elon- ton has covered the fibers and the cells appear to grow gation of the A. atlas fibers was similar to that of B. mori P r o p e r t i e s o f C o c o o n s a n d S i l k F i b e r s P r o d u c e d b y A t t a c u s a t l a s 23

Figure 8. Confocal laser scanning microscope image of the fibroblast cells on A. atlas fibers. The extensive spreading of F-actin is seen in red on the left image and the cell nuclei are seen in blue on the right image. silk fibers but much lower than that of the wild silks. 9. Kundu J, Dewan M, Ghoshal S, Kundu SC (2008) J Mater The A. atlas fibers showed considerably higher attach- Sci Mater Med 19:2679 ment and proliferation of cells than B. mori silk and ex- 10. Zhang Y (2000) Biotechnol Adv 20:91–100 tensive growth of F-actin demonstrating that the fibers 11. Tuskes PM, Tuttle JP, Collins MM (1996) The natural were biocompatible. Overall, the ability of the A. at- history of the saturniidae of the United States and Can- las to produce larger cocoons and silk fibers ada. Cornell University Press, New York with properties similar to that of B. mori silk offers an 12. Sen K, Babu MK (2004) J Appl Polym Sci 92:1080 opportunity to rear the A. atlas as alternative to the com- mon silks for commercial silk production. 13. Sen K, Babu MK (2004) J Appl Polym Sci 92:1098 14. Robson RM (1992) Silk: composition, structure and properties. In: Lewin M, Pearce EM (eds) Handbook Acknowledgments — The authors wish to thank the Agri- of fiber chemistry. Marcel Dekker Inc, New York, pp cultural Research Division at the University of Nebraska- 415–456 Lincoln, USDA Hatch Act and Multi-state Project S1026 for 15. Reddy N, Yang Y (2010) Int J Biol Macromol 46(4):419 their financial support to complete this research. Theau- 16. Reddy N, Yang Y (2010) J Mater Sci 45:4414 thors also thank Nathan Brockam with Reiman Gardens for 17. Reddy N, Yang Y (2010) J Mater Sci 45(24):6617 his help in collecting the A. atlas cocoons. 18. Nurmalitasari G, Kuroda F (2002) 4th international con- ference on wild silkmoths, April 23–26, Yogyakarta, References Indonesia 19. Razafimanantosoa T, Ravoahangimalala OR, Craig CL 1. Zhang X, Reagan MR, Kaplan D, Adv D (2009) Drug De- (2006) Madagascar Conserv Dev 1(1):34 liv Rev 61:988 20. Kakati LN, Chutia BC (2009) Trop Ecol 50(1):137 2. Mandal BB, Kundu SC (2010) Acta Biomater 6:360 21. Perez-Rigueiro J, Elices M, Lloraca J, Viney C (2002) J 3. Kasoju N, Bhonde RR, Bora U (2009) Mater Lett Appl Polym Sci 82:53 63(28):2466 22. Mondal M, Trivedy K, Kumar Caspian SN (2007) J Env 4. Kearns V, MacIntosh AC, Crawford A, Hatton PV (2008) Sci 5(2):63 Topics Tissue Eng 4:1 23. Lee Y (1999) Silk reeling and testing manual. FAO, 5. Reddy N, Yang Y (2011) Trends Biotechnol 29(10):490 Rome 6. Acharya C, Ghosh SK, Kundu SC (2008) J Mater Sci Ma- 24. Dash R, Mandal M, Ghosh SK, Kundu SC (2008) Acta ter Med 19:2827 Biomater 311:111 7. Acharya C, Ghosh SK, Kundu SC (2009) Acta Biomater 25. Padamwar MN, Pawar AP (2004) J Sci Ind Res 63:323 5(1):429 26. Poza P, Perez-Rigueiro J, Elices M, Llorca J (2002) Eng 8. Seidel A, Liivak O, Calve S, Adaska J, Ji G, Yang Z, Fract Mech 69:1035 Grubb D, Zax DB, Jelinski LW (2000) Macromolecules 33:775 Supplement R e d d y , Z h a o , & Y a n g i n J o u r n a l o f P o l y m e r s a n d t h e E n v i r o n m e n t 21 (2013)

Female Atlas (Attacus atlas) in the Wilhelma, Stuttgart, Germany. This is a live sitting high on the back wall of the insectarium laying . The wingspan is roughly 25 cm. http://en.wikipedia.org/wiki/File:Attacus_atlas_qtl1.jpg

Attacus atlas caterpillar. School of Ecology and Conservation, University of Agricultural Sciences Bangalore; http://commons.wikimedia.org/wiki/File:Attacus_atlas_cat.jpg